Immunoglobulins (also called antibodies) are glycoproteins, which specifically recognise foreign molecules. These recognised foreign molecules are called antigens. When antigens invade humans or animals, an immunological response is triggered which involves the production of antibodies by B-lymphocytes. By this immunological response, microorganisms, larger parasites, viruses and bacterial toxins can be rendered harmless. The unique ability of antibodies to specifically recognise and bind with high affinity to virtually any type of antigen, makes them useful molecules in medical and scientific research.
In vertebrates five immunoglobulin classes are described, including IgG, IgM, IgA, IgD and IgE, all of which differ in their function in the immune system. IgGs are the most abundant immunoglobulins in the blood. They have a basic structure of two identical heavy (H) chain polypeptides and two identical light (L) chain polypeptides. The H and L chains are kept together by disulfide bridges and non-covalent bonds. The chains themselves can be divided in variable and constant domains. The variable domains of the heavy and light chain (VH and VL) which are extremely variable in amino acid sequences are located at the N-terminal part of the antibody molecule. VH and VL together form the unique antigen-recognition site. The amino acid sequences of the remaining C-terminal domains are much less variable and are called CH1, CH2, CH3 and CL.
The non-antigen binding part of an antibody molecule is called the constant domain Fc and mediates several immunological functions, such as binding to receptors on target cells and complement fixation. The unique antigen-binding site of an antibody consists of the heavy and light chain variable domains (VH and VL). Each domain contains four framework regions (FR) and three regions called CDRs (complementarity determining regions) or hypervariable regions. The CDRs strongly vary in sequence and determine the specificity of the antibody. VL and VH domains together form a binding site, which binds a specific antigen.
Several functional antigen-binding antibody fragments could be engineered by proteolysis of antibodies, using for example papain digestion, pepsin digestions or other enzymatic approaches. Such a technique can be used to yield Fab, Fv or single domain fragments. Fab fragments are the antigen-binding domains of an antibody molecule. Fab fragments can be prepared by papain digestions of whole antibodies. Fv fragments are the minimal fragment (˜30 kDa) that still contains the whole antigen-binding site of a whole IgG antibody. Fv fragments are composed of both the variable heavy chain (VH) and variable light chain (VL) domains. This heterodimer, called Fv fragment (for fragment variable) is still capable of binding the antigen.
Heavy chain antibodies constitute about one fourth of the IgG antibodies produced by the camelids, e.g. camels and llamas (Hamers-Casterman C., et al. (1993)). These antibodies are formed by two heavy chains but are devoid of light chains. As a consequence, the variable antigen binding part is referred to as the VHH domain and it represents the smallest naturally occurring, intact, antigen-binding site, being only around 120 amino acids in length (Desmyter, A., et al. (2001)). Heavy chain antibodies with a high specificity and affinity can be generated against a variety of antigens through immunization (van der Linden, R. H., et al. (1999)) and the VHH portion can be readily cloned and expressed in yeast (Frenken, L. G. J., et al. (2000)). Their levels of expression, solubility and stability are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, M. A. et al. (1997)). Sharks have also been shown to have a single VH-like domain in their antibodies termed VNAR. (Nuttall et al. (2003)); (Dooley et al. (2003)); (Nuttall et al. (2004)).
Holt et al. (2003) reviews antigen-binding fragments called “domain antibodies” or dAbs which comprise only the VH or VL domain of an antibody and are consequently smaller than, for example, Fab and scFv. DAbs are the smallest known antigen-binding fragments of antibodies, ranging from 11 kDa to 15 kDa. They are highly expressed in microbial cell culture. Each dAb contains three of the six naturally occurring complementarity determining regions (CDRs) from an antibody.
The production of antibodies for use in conventional immunotherapy has been possible since the development of monoclonal antibody technology. This has led to the use of antibodies in many areas including research, medicine and recently in consumer applications.
Unfortunately, there are many problems associated with conventional immunotherapy. Such applications rely on the large scale production of antibodies and involve the use of the antibody or antibody fragment per se, that is the harvested protein from, for example, an antibody expression system. Thus, the associated production costs, including the requirement for purification of the antibodies before administration, are expensive and make their wide scale application as immunotherapeutics prohibitive. Furthermore, in order to treat a large part of the population, large amounts of conventional immunotherapy products would be required. If, for example, the therapy was based on colostrum and/or immunised chicken eggs, it would be difficult to provide the required amounts of the immunotherapy products in a large volume.
Furthermore, in a recent article, “In situ delivery of passive immunity by lactobacilli producing single-chain antibodies” Nature Biotechnol. (2002) 20, 702-706, Kruger et al reported on the production of scFv antibody fragments against Streptococcus mutans by the Gram positive food grade bacteria Lactobacillus zeae. This treatment involved in situ delivery of passive immunity at oral mucosal sites only wherein the single chain antibody fragments were shown to deliver protection against dental caries in rats.
Lactobacilli have been investigated with regards to their anti-diarrhoeal properties since the 1960's (Beck, C., et al. Beneficial effects of administration of Lactobacillus acidophilus in diarrhoeal and other intestinal disorders. Am. J. Gastroenterol (1961) 35, 522-30). A limited number of recent controlled trials have shown that certain strains of lactobacilli may have therapeutic as well as prophylactic properties in acute viral gastroenteritis (Mastretta, E., et al. Effect of Lactobacillus CG and breast-feeding in the prevention of rotavirus nosocomial infection. J. Pediatr. Gastroenterol. Nutr. (2002) 35, 527-531). Selected strains of Lactobacillus casei and Lactobacillus plantarum have also been shown to exert strong adjuvant effects on the mucosal and the systemic immune response. Lactobacilli are well-known bacteria applied in the production of food products. For example yogurt is normally made by fermenting milk with among others a Lactobacillus strain. The fermented acidified product, still containing the viable Lactobacillus, is then cooled and consumed at the desired moment.
Another application of Lactobacillus in food products is in the production of meat products for example sausages. Here the Lactobacillus is added to the meat mass prior to applying the casing, followed by a period of ripening in which the fermentation process takes place.
Still another application of Lactobacillus in the production of food products is the brining of vegetables such as cabbage (sauerkraut), carrots, olives or beets. Here the natural fermentation process can be controlled by the addition of an appropriate Lactobacillus starter culture.
The application of Lactobacillus in food products is often associated with several health effects, see for example A. C. Ouwehand et al. in Int. Dairy Journal 8 (1998) 749-758. In particular the application of products is associated with several health effects for example relating to gut well being such as IBS (Irritable Bowel Syndrome), reduction of lactose maldigestion, clinical symptoms of diarrhoea, immune stimulation, anti-tumour activity and enhancement of mineral uptake.
WO 99/23221 discloses multivalent antigen binding proteins for inactivating phages. The hosts may be lactic acid bacteria which are used to produce antibody binding fragments which are then harvested and used. WO 99/23211 discloses adding the harvested antibody fragments to provide anti-diarrhea effects.
WO 00/65057 is directed to the inhibition of viral infection using monovalent antigen-binding proteins. The antigen-binding protein may be a heavy chain variable domain derived from an immunoglobulin naturally devoid of light chains, such as those derived from Camelids as described in WO 94/04678. WO 00/65057 discloses transforming a host with a gene encoding the monovalent antigen-binding proteins. Suitable hosts can include lactic acid bacteria. This disclosure relates to the field of fermentation processing and the problem of phage infection which hampers fermentation. Specifically, llama VHH fragments per se are used to solve the problem of phage infection by neutralising Lactoccoccus lactis bacteriophage P2.
Both WO 00/65057 and WO 99/23221 involve the use of antibody fragments harvested from a bacterial expression system.
U.S. Pat. No. 6,605,286 is directed to the use of gram positive bacteria to deliver biologically active polypeptides, such as cytokines, to the body. U.S. Pat. No. 6,190,662 and EP 0 848 756 B1 are directed to methods for obtaining surface expression of a desired protein or polypeptide. Monedero et al 2004 is directed to in-vitro studies on the use of single-chain antibodies (scFv) expressed by L. casei which recognise the VP8 and fraction of rotavirus outer capsid and blocks rotavirus infection in vitro. However, none of these documents disclose the use of heavy chain immunoglobulins or fragments of the VHH or VNAR type or domain antibodies.
One major disadvantage of these known systems is that the use of antibodies or antibody fragments per se (i.e. a harvested protein) in the treatment of a disease in a human may result in the antibody being degraded or digested before they provide the desired health benefits and even before they reach the desired location. Furthermore, it is often desirable to ensure that the antibody or antibody fragments are active in a specific region of the body. This will depend on the particular infection being treated.
Enteropathogenic micro-organisms are micro-organisms which can produce disease in the intestinal tract. Examples of such micro-organisms include E. coli and Salmonella. 
Another example of an enteropathogen is rotavirus. Rotavirus continues to be the single most common cause of infantile diarrhoea in the world and most children get infected during the first 5 years of life. In developing countries, rotavirus induced diarrhoea may cause 600,000 to 870,000 deaths each year and in developed countries, rotavirus disease accounts for immense economic loss. Following the intussusception related withdrawal of the Rhesus rotavirus-tetravalent vaccine in July 1999, several strategies have been employed to try to develop a safe vaccine. Five oral live attenuated rotavirus vaccines are presently undergoing clinical trials for use in children, for example Barnes, G. L. et al. (1997). However, in particular in very young, malnourished children in developing countries the efficacy of such vaccinations might be limited. Several studies have also been conducted using passive immunotherapy, for example Offit, P. A. et al. (1985).
Secretory immunoglobulins are implicated as a first line of defense against many mucosal pathogens, including rotavirus infection. Protection from clinical disease appears to rely mainly on the production of neutralizing antibodies against the outer capsid proteins VP4 or VP7 (Ruggeri, F. M. at al. (1998)); (Giammarioli, A-M. et al. (1996)). Recently however, non-neutralizing VP6 specific IgA antibodies have also been shown to inhibit rotavirus replication (Feng, N. et al. (2002)); Schwartz-Cornil, I. et al. (2002)). Both hyperimmune bovine colostrum and chicken egg yolks derived antibodies have previously been shown to be effective in the treatment of rotavirus diarrhoea (Davidson et al. (1989)); (Sarker, S. A., et al. (2001)). Successful treatment of rotavirus diarrhoea in children with immunoglobulin from immunized bovine colostrums has also been demonstrated (Sarker S. A. et al. (1998)). However, the high costs of production of these immunoglobulin preparations make their wide scale application as immunotherapeutics prohibitive.
To date, no specific therapy is available for the widespread management of enteropathogenic micro-organisms and viruses. For example, the current management of rotavirus induced diarrhoea mainly involves prevention and oral rehydration. Thus, it is desirable to provide an alternative means for the management of enteropathogenic micro-organisms, by therapy and/or prophylaxis.
The present invention is directed to a new method of therapy and/or prophylaxis of enteropathogenic micro-organisms.